The first objective of the proposed work is to study the catalytic mechanism of glyceraldehyde-3-phosphate dehydrogenase employing the techniques of electron paramagnetic resonance (EPR) and saturation transfer-EPR (ST-EPR) spectroscopies. With the use of various spin-labeled probes, different domains within the catalytic center will be characterized. These EPR and ST-EPR studies will be correlated with the crystallographic analyses and amino acid sequence work of other laboratories. The EPR work on enzymatic catalysis will be extrapolated into the larger framework of muscle contraction and relaxation. The second objective is to synthesize isotopically labeled (2H, 14C, 15N) spin probes and a variety of new spin labeled analogues which can be used for determining the geometry of the catalytic center of enzymes, characterizing subunit interactions, and describing the motional properties of membrane bound enzymes. The rationale and design of a number of spin labels as well as the synthetic procedures are discussed. All new spin labels and methodology developed for this GAPDH study will be of great utility in the study of enzyme mechanisms and membrane-protein interaction in general.